Nearly one-third of all human breast cancers, including most of the highly aggressive comedo- and inflammatory-type breast cancers, over express the neu/c-erbB-2 oncogene product p185 on their plasma membranes. The goal of the proposed work is to develop radioligands targeted to the p185 receptor. The hypothesis is that scintigraphic imaging can detect tumor-associated p185 accurately and noninvasively and, thereby, improve the accuracy of breast cancer staging and surveillance in the subset of patients whose tumors over express p185. Although monoclonal antibodies (mAb) reactive with the human p185 receptor are available, tumor imaging with radiolabeled Mab has been largely disappointing; tumor localization is poor and background activity is high. Intact Mab fail as site-directed biological probes because of: 1) large size (150 kDa); 2) non-specific binding; 3) slow plasma clearance rate; and 4) immunogenicity. To circumvent these limitations, the investigators propose to target the p185 receptor using newly-developed antibody-derived small molecules (less than 2 Kda) that mimic the antibody complementarily determining region (CDR). Dr. Mark Greene and his co-workers developed a conformationally constrained, aromatically-modified peptide analogue based on the heavy chain CDR3 of 4D5, a murine mAb reactive with human p185. This CDR analogue is called 4D5.AME, where AME stands for Aromatic Modified Exocyclic. Preliminary studies with 4D5.AME demonstrate excellent binding affinity and exceptional bioactivity against human breast cancers that over express p185. Thus, 4D5.AME is a potentially superb site-directed biological probe for delivery of gamma-emitting radionuclides to tumor-associated p185. Accordingly, the specific aims are to: 1) radiolabel 4D5.AME with iodine-123, technetium-99m and indium-111; 2) determine the stability, affinity, and specificity of 4D5.AME radioligands in vitro using human breast cancer cell lines; 3) determine the redistribution and plasma clearance rates of 4D5.AME radioligands in vivo using a xenograft tumor model in nude mice; and 4) perform, optimize, and evaluate planar scintigraphic imaging of 4D5.AME radioligands in tumor-bearing mice. The proposed imaging agents incorporate significant design improvements compared with existing agents. However, it is impossible to predict whether radiolabeling will disturb critical residues in the 4D5.AME receptor binding site. Any promising 4D5.AME radioligands developed during this R21 granting period will be used as preliminary data in support of a future R01 proposal.